Advances in methods of modern drug discovery such as those utilizing high throughput screening assays of combinatorial chemical libraries continue to provide us with unprecedented numbers of drug candidates for treatment of diseases. However, the problem of drug toxicity to the host system continues to be a significant rate-limiting step in the translation of drug candidates from the laboratory to the healthcare setting. Similar toxicity problems hinder the development of industrial and household chemicals as well. Besides being expensive and time-consuming, currently available toxicological screening assays fail to detect all toxicities associated with human therapy. For instance, as much as one third of all prospective human therapeutics fail in the first phase of human clinical trials because of unexpected toxicity. Better means of screening potential therapeutics for potential toxicity would reduce the cost and uncertainty of developing new therapeutics and, by reducing uncertainty, would encourage the private sector to commit additional resources to drug development.
Currently available alternatives to traditional “single-reporter” cell lines and animal toxicity testing do not fully meet these needs. For example, Farr, U.S. Pat. No. 5,811,231, provides methods of identifying and characterizing toxic compounds by choosing selected stress promoters and determining the level of the transcription of genes linked to these promoters in cells of various cell lines. This method therefore depends on the degree to which both the promoter and the cell lines are representative of the effect of the potentially toxic agent on the organism of interest.
The use of hybridization arrays of oligonucleotides provides another route for determining the potential toxicity of chemical compositions. Exposing cells of a culture to a chemical composition and then comparing the expression pattern of the exposed cells to that of cells exposed to other chemical agents permits one to detect patterns of expression similar to that of the test compound, and thus to predict that the toxicities of the chemical compositions will be similar. See, e.g., Service, R., Science (1998), 282:396-399. These methods suffer from the fact that individual cell lines may not be fully representative of the complex biology of an intact organism. Moreover, even repeating the tests in multiple cell lines does not reproduce or account for the complex interactions among cells and tissues that occur in an organism.
Liver cell-based toxicity assays are also known. For example, Maier describes development of an in vitro toxicity test with cultures of freshly isolated rat hepatocytes. Maier, P., Experientia (1988), 44(10):807-817. This test is based on drug-induced pathological alterations in ploidy in hepatocytes as indicators of compounds which interfere with cell differentiation in liver. Sawai and Awata describe a method for culturing liver cells that can be used for testing the toxicity of test substances. Sawai and Awata, Japanese Patent 10179150. Takashina and Naoki describe established subculturable hepatic cells obtained by fusing a subculturable hepatic cell strain to a hepatocyte that can be used for toxicity tests. Takashina and Naoki, Japanese Patent 06319535. Again, toxicity assays using cell lines such as these may not fully take into account the complex biology of an intact organism or tissue, and cannot address the contributions of cell and tissue interactions in determining toxicity effects.
Lockhart et al. describe a method of screening a drug for deleterious side effects on a cell using expression profiles of a group of known genes. Lockhart et al., U.S. Pat. No. 6,033,860. This method assesses alterations in expression of 16 known genes, and therefore is limited to only drug and toxicity types that alter the expression of a very small number of genes whose identity is known and expression level can be specifically measured.
A method for identifying or testing cytotoxicity of an agent based on expression of cytochrome P450 is also known. Harris et al., U.S. Pat. No. 5,660,986. This method is based on testing cytotoxicity of agents on human bronchial and liver epithelial cell lines expressing exogenous cytochrome P450. This method is limited by its narrow focus on the expression of a particular gene and its nature as an assay that is based on cell lines that do not take account of complex cell and tissue interactions.
Largely because it performs a vital role in detoxifying poisonous substances in the body, the liver is the site wherein the toxicity effects of substances are most frequently and severely manifested. See, e.g., Table 1. Therefore, toxicity assays that focus primarily on the effects of potentially toxic compounds on cells related to or found in the liver would be expected to be particularly informative.
What is needed in the art is a method of systematically testing chemical compositions for potential toxicity in a milieu in which cells interact with cells of other types and that is most relevant to the common target(s) of toxic effects. What is further needed is a means of doing so which is relevant to the effect of the composition on whole organisms or tissues, without the cost, time, and ethical ramification of animal and human testing. The present invention addresses these and other needs.